Aldose and aldehyde reductase are closely related members of the aldo-keto reductase superfamily. Aldose reductase is implicated in the pathogenesis of diabetic cataracts, retinopathy, neuropathy and nephropathy. Aldose reductase inhibitors, proposed for the prevention and therapy of those diabetic complications, are not specific and inhibit both enzymes as well as other members of the superfamily. The objectives of this proposal are to compare the atomic structure and the catalytic and inhibitory mechanisms of these two enzymes so as to obtain a better understanding of the enzymatic mechanism(s) in order to rationally develop highly specific inhibitors unique to each enzyme, as well as to better understand the normal operation of aldose reductase in the whole organism. It is proposed to: (1) investigate the joint role of the NADP-binding and C-terminal loops of aldose reductase in the regulation of catalysis and co-factor exchange, and particularly, the potential role of cysteine 298 in the thiol-mediated regulation of the enzyme; (2) determine the mechanism, structure, and inhibition of human aldehyde reductase in comparison to aldose reductase; (3) use the kinetic and crystallographic information obtained to design specific inhibitors for aldose and aldehyde reductase, respectively, using organic solvent mapping of enzyme surface, proprietary peptidomimetic compounds and knowledge of the mechanistic pathway; and (4) determine the basis for the significant decrease in activity of murine recombinant wild-type aldose reductase in preparation for future testing of the role of aldose reductase in diabetic retinopathy and other complications by introduction of appropriate mutations into the mouse germ-line.